Pkc activators and anticoagulant in regimen for treating stroke

ABSTRACT

The present disclosure provides a method for treating stroke by administering to a subject an anticoagulant, e.g., recombinant tissue plasminogen activator (rTPA), and a protein kinase C (PKC) activator followed by adminstration of at least one PKC activator for a duration of treatment. The methods disclosed herein may limit the size of infarction and/or reduce mortality, the disruption of the blood-brain barrier, and/or the hemorrhagic damage due to ischemic stroke compared with rTPA administration alone; and may also extend the therapeutic time window for administering rTPA after a stroke. Also disclosed are kits comprising rTPA and a PKC activator for treating stroke.

This application claims priority to U.S. Provisional Application Nos.61/362,464 filed Jul. 8, 2010, 61/412,753 filed Nov. 11, 2010, and61/412,747 filed Nov. 11, 2010, the entire disclosures of which areincorporated by reference herein.

The present disclosure relates generally to administration of ananticoagulant, e.g., recombinant tissue plasminogen activator (rTPA),and a protein kinase C (PKC) activator followed by administering atleast one PKC activator for a duration of treatment to treat a subjectfollowing ischemic stroke. The methods disclosed herein may limit thesize of infarction and/or reduce mortality, the disruption of theblood-brain barrier, and/or the hemorrhagic damage due to ischemicstroke compared with rTPA administration alone. The methods disclosedherein may also extend the therapeutic window in which rTPA can beadministered following a stroke and still be efficacious. Compositionsand kits comprising rTPA and a PKC activator are also disclosed.

Stroke

Stroke, also known as a cerebrovascular accident (CVA), is a medicalemergency and can cause permanent neurologic damage or even death if notpromptly diagnosed and treated. It is the third leading cause of deathand the leading cause of adult disability in the United States andindustrialized European nations. On average, a stroke occurs every 45seconds and someone dies every 3 minutes. Of every 5 deaths from stroke,2 occur in men and 3 in women.

A stroke is an acute neurological injury in which the blood supply to apart of the brain is interrupted, leading to the sudden loss of neuronalfunction. The blood supply to the brain may be interrupted in severalways; the disturbance in perfusion is commonly arterial, but may bevenous.

Different types of stroke include ischemic stroke and hemorrhagicstroke. Ischemic stroke or cerebral ischemia is caused by a temporary orpermanent restriction of cerebral blood flow and oxygen supply causedby, for example, an embolis (embolic stroke) or blood clot (thrombolyicstroke). In contrast, a hemorrhagic stroke is caused by the blood vesselrupture (e.g., ruptured aneurysm), which leads to severe bleeding in thebrain.

In stroke, the part of the brain with disturbed perfusion no longerreceives adequate oxygen (hypoxia). This initiates the ischemic cascadewhich causes brain cells to die or be seriously damaged, impairing localbrain function. A transient ischemic attack (TIA) or “mini-stroke”normally lasts less than 24 hours, but is associated with the samesymptoms as stroke such as sudden numbness or weakness of the face, arm,or leg; sudden confusion, trouble speaking or understanding; suddentrouble seeing in one or both eyes; and/or sudden trouble walking,dizziness, loss of balance or coordination. Typically, TIAs do notresult in permanent brain injury through acute infarction (i.e., tissuedeath) but they may indicate serious risk of subsequent stroke. Aninfarctive stroke typically involves a more severe vessel blockage thatcan last longer than 24 hours without intervention. Cerebral infarctionsvary in severity; about one third of the cases result in death.

Ischemia may be confined to a specific region of the brain (focalischemia), or may affect large areas of brain tissue (global ischemia).Significant brain injury can occur after the immediate ischemic event.Neuronal death and injury after cerebral ischemia involve pathologicalchanges associated with necrosis and delayed apoptosis. Neurons in theinfarction core of focal, severe stroke are immediately dead and cannotbe saved by pharmacologic intervention. The ischemic penumbra,consisting of the brain tissue around the core in focal ischemic stroke,and the sensitive neurons/network in global cerebral ischemia, however,are maintained by a diminished blood supply. The damage to thispenumbral brain tissue occurs in a “delayed” manner, starting 4-6 hoursas the second phase or days and weeks later as the so-called thirdphase, after ischemic stroke.

A consistent consequence of cerebral ischemia/hypoxia in humans andother mammals is central nervous system dysfunction, the nature of whichdepends on the location and extent of injury. Global cerebralischemia/hypoxia selectively injures or damages the pyramidal neurons inthe dorsal hippocampal CA1 area, which are essential for episodicmemory, providing a sensitive measure for monitoring ischemic damage andrecovery functionally. After a cerebral ischemia of about 15 minutes,for example, the hippocam pal CA1 pyramidal cells start to degeneratewithin 2-3 days, and reach the maximal extent of cell death a week afterthe ischemic event. The sensitive neuronal structures in global cerebralischemia and the ischemic penumbra are “at-risk” tissues. Their salvagethrough intervention or further damage in the subsequent days or weeksdetermine dramatic differences in long-term disability.

Following ischemic stroke, there is a transient loss of blood-brainbarrier (BBB) function that happens within minutes or hours of the eventas the interruption in blood flow and lack of oxygen leads to increasedBBB permeability. DiNapoli et al., Neurobiology of Aging (2008) vol. 29,pp. 753-764. Disruption of the BBB, in turn, results in loss of ionichomeostasis and loss of neurotransmitter homeostasis. Immune cells andtoxic compounds can enter the brain during that period, providing anadded neurotoxic insult. Edema can form during the early stages ofischemia with a rate related to the rate of sodium transport from bloodto brain, i.e., increased sodium transport across the BBB contributes tocerebral edema formation. Betz and Coester, Stroke (1990), vol. 21, pp.1199-1204. Thus, measurements of both edema and ion uptake in the brainare indicators of brain pathology following stroke. The loss ofintegrity of the barrier could lead to adverse hemorrhages as aconsequence of thrombolytic therapy, e.g., administration of recombinanttissue plasminogen activator (rTPA). Tanne et al., Nature ReviewsNeurology (2008), vol. 4, pp. 644-645.

Despite the medical emergency presented by stroke, and preclinicalstudies suggesting agents that may be effective in arresting thepathological processes involved, options for treating stroke remainlimited. The main treatment available is rTPA, a thrombolytic agent andthe only drug currently approved by the U.S. Food and DrugAdministration for acute/urgent treatment of ischemic stroke. The rTPAprotein is an enzyme (serine protease) that initiates local fibrinolysisvia fibrin-enhanced conversion of plasminogen to plasmin. rTPA is usedto improve neurologic recovery and reduce the incidence of disability.Experimental models of stroke use rTPA, for example, in reperfusionafter inducing focal embolic ischemia via middle cerebral arteryocclusion (MCAO). DiNapoli et al., J. Neurosci Methods (2006), vol. 154,pp. 233-238.

The effectiveness of rTPA and other potential agents for arrestinginfarct development depends on early administration or even before theischemic event, if possible. Treatment with rTPA is designed to achieveearly arterial recanalization such that rTPA must be administered within3 hours after the event to be effective. This time dependency limits itsclinical usefulness; the narrow therapeutic time window and exclusioncriteria in treating ischemic stroke leads to about only 5% of candidatepatients receiving effective intravenous thrombolytic therapy. Forexample, one study reported 13% mortality at 30 days after an acuteischemic stroke, with more than two thirds of the deaths related to theinitial stroke. Nedeltcheva et al., Swiss Med. Wkly (2010), vol. 140,pp. 254-259. The recommended dose of rTPA is 0.9 mg/kg (maximum dose 90mg) where 10% is given by rapid (˜1 min.) Iv injection and the remainderby constant infusion over 60 min. No aspirin, heparin, or warfarinshould be administered for 24 hours following rTPA. rTPA is sold underthe names alteplase (Activase®) and streptokinase (Streptase®).

Use of rTPA following stroke is controversial because it carries anincreased risk of intracranial hemorrhage, reperfusion injury, anddiminishing cerebral artery reactivity. Thus, rTPA is should not beadministered to treat hemorrhagic stroke. Unfortunately, it may not beimmediately apparent whether a patient suffered an ischemic orhemorrhagic stroke, which further limits the usefulness of rTPA withinits limited therapeutic time window. In addition, hemorrhagictransformation can spontaneously follow ischemic stroke. For example,one study found that 6.4% of patients with large strokes developedsubstantial brain hemorrhage as a complication from being given rTPA.The National Institute of Neurological Disorders and Stroke rt-PA StrokeStudy Group, N. Engl. J. Med. (1995), vol. 333, pp. 1581-1587.

rTPA is contraindicated or advised against in the following patientpopulations:

Evidence of intracranial hemorrhage on pretreatment CT scan

Clinical presentation suggestive of subarachnoid hemorrhage, even withnormal CT scan

Active internal bleeding

Known bleeding diathesis, including but not limited to: having aplatelet count<100,000/mm; receiving heparin within 48 hours and havingan elevated activated partial thromboplastin (aPTT) greater than upperlimit of normal for laboratory; and current use of oral anticoagulants(e.g., warfarin sodium) or recent use with an elevated prothrombintime>15 seconds

Within 3 months any intracranial surgery, serious head trauma, orprevious stroke

History of gastrointestinal or urinary tract hemorrhage within 21 days

Recent arterial puncture at a noncompressible site

Recent lumbar puncture

On repeated measurements, systolic blood pressure greater than 185 mm Hgor diastolic blood pressure greater than 110 mm Hg at the time treatmentis to begin, and patients requiring aggressive treatment to reduce bloodpressure to within these limits.

History of intracranial hemorrhage

Abnormal blood glucose (<50 mg/dL or >400 mg/dL)

Post myocardial infarction pericarditis

Patient observed to have seizure at the same time the onset of strokesymptoms were observed

Known arteriovenous malformation, or aneurysm

See, e.g., TPA Stroke Study Group Guidelines, The Brian Attack Coalition(available athttp://www.stroke-site.org/guidelines/tpa_guidelines.html).

Studies have suggested an association between hematocrit, reducedreperfusion and greater infarct size, and between elevated hemoglobinlevels and increased rates of all-cause death. Tanne et al., BMCNeurology (2010), vol. 10:22, pp. 1-7. Elevated levels of glycatedhemoglobin (HbA1c) increases the risk of heart attacks and strokes indiabetic patients. Glycated hemoglobin, even at levels considered in thenormal range, can also be an independent predictor of ischemic stroke innon-diabetic adults. Selvin et al., N. Engl. J. Med. (2010), vol. 362,pp. 800-811. Elevated hemoglobin may also increase the risk of stroke inpatients with chronic kidney disease.

Low hemoglobin levels (e.g., levels>6.0% or 8.8 g/dL, anemia) have alsobeen identified as a risk factor for ischemic stroke, especiallyfollowing cardiac surgery. In addition, anemia can worsen brain ischemiafollowing acute ischemic stroke, and is associated with a poor prognosisand increased mortality after one year compared with non-anemic strokepatients (hemoglobin<13 g/dL in males, <12 g/dL in women). Tanne et al.,BMC Neurology (2010), 10:22. Studies have also reported that childrenwith sickle cell anemia have an increased stroke risk.

Protein Kinase C

Protein kinase C (PKC) is one of the largest gene families ofnon-receptor serine-threonine protein kinases. Since the discovery ofPKC in the early eighties and its identification as a major receptor forphorbol esters, a multitude of physiological signaling mechanisms havebeen ascribed to this enzyme. Kikkawa et al., J. Biol. Chem. (1982),vol. 257, pp. 13341-13348; Ashendel et al., Cancer Res. (1983), vol. 43:4333-4337. The interest in PKC stems from its unique ability to beactivated in vitro by calcium and diacylglycerol (and phorbol estermimetics), an effector whose formation is coupled to phospholipidturnover by the action of growth and differentiation factors. Activationof PKC involves binding of 1,2-diacylglycerol (DAG) and/or1,2-diacyl-sn-glycero-3-phospho-L-serine (phosphatidyl-L-serine, PS) atdifferent binding sites. An alternative approach to activating PKCdirectly is through indirect PKC activation, e.g., by activatingphospholipases such as phospholipase Cγ, by stimulating the Ser/Thrkinase Akt by way of phosphatidylinositol 3-kinase (PI3K), or byincreasing the levels of DAG, the endogenous activator. Nelson et al.,Trends in Biochem. Sci. (2009) vol. 34, pp. 136-145. Diacylglycerolkinase inhibitors, for example, may enhance the levels of the endogenousligand diacylglycerol, thereby producing activation of PKC. Meinhardt etal., Anti-Cancer Drugs (2002), vol. 13, pp. 725-733. Phorbol esters arenot suitable compounds for eventual drug development because of theirtumor promotion activity. Ibarreta et al. Neuroreport (1999), vol. 10,pp. 1035-1040).

The PKC gene family consists of 11 genes, which are divided into foursubgroups: (1) classical PKC α, β1, β2 (β1 and β2 are alternativelyspliced forms of the same gene) and γ; (2) novel PKC δ, ε, η, and θ; (3)atypical PKC ζ and ι/λ; and (4) PKC μ. PKC μ resembles the novel PKCisoforms but differs by having a putative transmembrane domain. Blobe etal. Cancer Metastasis Rev. (1994), vol. 13, pp. 411-431; Hug et al.Biochem. J. (1993) vol. 291, pp. 329-343; Kikkawa et al. Ann. Rev.Biochem. (1989), vol. 58, pp. 31-44. The classical PKC isoforms α, β1,β2, and γ are Ca²⁺, phospholipid, and diacylglycerol-dependent whereasthe other isoforms are activated by phospholipid, diacylglycerol but arenot dependent on Ca²⁺ and no activator may be necessary. All isoformsencompass 5 variable (VI-V5) regions, and the α, β, and γ isoformscontain four (C1-C4) structural domains which are highly conserved. Allisoforms except PKC α, β, and γ lack the C2 domain, the ι/λ and ηisoforms also lack nine of two cysteine-rich zinc finger domains in C1to which diacylglycerol binds. The C1 domain also contains thepseudosubstrate sequence which is highly conserved among all isoforms,and which serves an autoregulatory function by blocking thesubstrate-binding site to produce an inactive conformation of theenzyme. House et al., Science (1987), vol. 238, pp. 1726-1728.

Because of these structural features, diverse PKC isoforms are thoughtto have highly specialized roles in signal transduction in response tophysiological stimuli as well as in neoplastic transformation anddifferentiation. Nishizuka, Cancer(1989), vol. 10, pp. 1892-1903;Glazer, pp. 171-198 in Protein Kinase C, I. F. Kuo, ed., Oxford U.Press, 1994. For a discussion of PKC modulators see, for example,International Application No. PCT/US97/08141 (WO 97/43268) and U.S. Pat.Nos. 5,652,232; 6,080,784; 5,891,906; 5,962,498; 5,955,501; 5,891,870and 5,962,504, each incorporated by reference herein in its entirety.

Fatty Acids as PKC Activators

Some polyunsaturated fatty acids (PUFAs) such as arachidonic acid(5,8,11,14-eicosatetraenoic acid) are known natural activators of PKC.Docosahexaenoic acid (DHA) (all-cis-docosa-4,7,10,13,16,19-hexaenoicacid), for example, is a PKC activator and has been shown to slow theaccumulation of Aβ and tau proteins associated with brain-cloggingplaques and tangles implicated in Alzheimer's disease. Sahlin et al.,Eur. J. Neurosci. (2007), vol. 26, pp. 882-889. Some PUFA derivativesalso have reported PKC activity. Kanno et al., J. Lipid Res. (2007),vol. 47, pp. 1146-1156.

Problems associated with use of PUFAs as PKC activators include a needfor high concentrations to achieve effects, non-specific activation ofPKC isoforms, and rapid metabolism and sequestration of unmodified PUFAsinto fat tissues and other organs where they are incorporated intotriglycerides and chylomicrons. Ishiguro et al., J. Pharmacobiodyn(1988) vol. 11, pp. 251-261. PUFAs may also cause adverse side effects.For example, arachidonic acid is a biochemical precursor toprostaglandins, thromboxanes, and leukotrienes, which have potentpro-inflammatory effects. This may be undesirable for treatment of somediseases like Alzheimer's disease, whose pathology likely involvesinflammation. Other essential fatty acids may also cause biologicaleffects such as enhancing nitric oxide signaling, anti-inflammatoryeffects, and inhibition of HMG-CoA reductase, which could interfere withcholesterol biosynthesis.

The activation of PKC has been shown to improve learning and memory.See, e.g., Hongpaisan et al., Proc. Natl. Acad. Sci. (2007) vol. 104,pp. 19571-19578; International Application Nos. PCT/US2003/007101 (WO2003/075850); PCT/US2003/020820 (WO 2004/004641); PCT/US2005/028522 (WO2006/031337); PCT/US2006/029110 (WO 2007/016202); PCT/US2007/002454 (WO2008/013573); PCT/US2008/001755 (WO 2008/100449); PCT/US2008/006158 (WO2008/143880); PCT/US2009/051927 (WO 2010/014585); and PCT/US2011/000315;and U.S. application Ser. Nos. 12/068,732; 10/167,491 (now U.S. Pat. No.6,825,229); Ser. Nos. 12/851,222; 11/802,723; 12/068,742; and12/510,681; each incorporated by reference herein in its entirety. PKCactivators have been used to treat memory and learning deficits inducedby stroke upon administration 24 hours or more after inducing globalcerebral ischemia through two-vessel occlusion combined with a shortterm (˜14 minutes) systemic hypoxia. Sun et al., Proc. Natl. Acad. Sci.(2008) vol. 105, pp. 13620-13625; Sun et al., Proc. Natl. Acad. Sci.(2009) vol. 106, pp. 14676-14680.

The present disclosure relates to a method of treating a subject who hassuffered an ischemic event comprising: (a) administering to the subjectan anticoagulant and at least one protein kinase C (PKC) activatorwithin about 24 hours after the ischemic event; and (b) administering atleast one PKC activator after step (a) for a duration of treatment;wherein the PKC activators of step (a) and step (b) are the same ordifferent.

The present disclosure further relates to a method of treating stroke ina subject in need thereof comprising: (a) identifying a subject havingsuffered a stroke; (b) administering to the subject atherapeutically-effective amount of a protein kinase C (PKC) activator;(c) determining whether the subject suffered an ischemic stroke orhemorrhagic stroke; (d) if the subject suffered an ischemic stroke,administering a therapeutically-effective amount of an anticoagulant;and (e) administering at least one PKC activator for a duration oftreatment; wherein the PKC activators of step (b) and step (e) are thesame or different.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a the amount of hemoglobin the ipsilateral andcontralateral cortices following ischemic stroke in rats treated witheither rTPA at 6 hours following stroke, or a combination ofbryostatin-1 administered 2 hours after the stroke, followed 6 hourslater by rTPA.

FIG. 2 shows a the percent of brain edema following ischemic stroke inrats treated with either rTPA at 6 hours following the stroke, or acombination of bryostatin-1 administered 2 hours after the stroke,followed 6 hours later by rTPA.

FIG. 3 shows the results of uptake of Evans Blue dye in the ipsilateraland contralateral cortices in rats treated with rTPA 2 hours followingthe stroke, or a combination of rTPA at 2 hours followed 6 hours laterwith bryostatin-1.

FIG. 4 shows the results of sodium fluoride uptake in the ipsilateraland contralateral cortices in rats treated with rTPA 2 hours followingthe stroke, or a combination of rTPA at 2 hours followed 6 hours laterwith bryostatin-1.

FIG. 5 shows the structures of various fatty acid derivatives accordingto the present disclosure (BR-101 through BR-118).

FIG. 6 shows PKCε activation by BR-101 (DCP-LA), BR-102, and BR-103.

FIG. 7 shows PKCε activation by vanous concentrations of BR-111 (DHA-CP6methyl ester), BR-114 (EPA-CP5 ester), and BR-115 (AA-CP4 methyl ester).

FIG. 8 shows PKCε activation by various concentrations ofcyclopropanated and epoxidized fatty acid methyl esters: cyclopropanatedlinolenyl alcohol (BR-104); cyclopropanated linoleyl alcohol (BR-105);epoxystearic acid (BR-116); vernolic acid methyl ester (BR-117); andcyclopropanated vernolic acid methyl ester (BR-109).

FIG. 9 shows PKC activation over time by various concentrations ofbryostatin in H19-7/IGF-IR rat hippocampal neurons.

FIG. 10 shows PKC activation over time by bryostatin and DCP-LA in rathippocampal primary neurons.

FIGS. 11a and 11b show levels of intracellular (FIG. 11a ) and secreted(FIG. 11b ) Aβ in neuro2a (N2A) cells exposed to bryostatin, BR-101(DCP-LA), and BR-111 (DHA-CP6).

FIG. 12 shows the effect of BR-111 (DHA-CP6) (0.1 μM to 10 μM) ondegradation of exogenously applied Aβ in SH-SY5Y neuroblastoma cells.

FIGS. 13a, 13b and 13c show effects of (FIG. 13a ) bryostatin, BR-101(DCP-LA) and BR-111 (DHA-CP6) on TACE activity in N2a neuroblastomacells transfected with human APPSwe/PS1D; (FIG. 13b ) variousconcentrations of bryostatin on TACE activity in rat cortical primaryneurons; and (FIG. 13c ) BR-111 (DHA-CP6) on TACE activity in ratcortical primary neurons.

FIG. 14 shows activation of endothelin converting enzyme (ECE) bybryostatin (0.27 nM), BR-101 (DCP-LA) (1 μM), BR-111 (DHA-CP6) (1 μM),and ethanol in SH-SY5Y neuroblastoma cells.

FIG. 15 shows effects of BR-101 (DCP-LA) and BR-111 (DHA-CP6) (1-100 μM)on cell survival and cell proliferation in SH-SY5Y neuroblastoma cells.

FIG. 16 shows a spatial water maze performance of rats over trainingtrials. Data are shown as means±SEM. Bry, bryostatin-1; Isch, cerebralischemia; MCDA, 4-methylcatechol-diacetic acid.

FIG. 17 shows target quadrant ratio during probe test. Bry,bryostatin-1; Isch, ischemia; MCDA, 4-methylcatechol-diacetic acid *:p<0.05. NS: p>0.05.

DETAILED DESCRIPTION

Particular aspects of the disclosure are described in greater detailbelow. The terms and definitions as used in the present application andas clarified herein are intended to represent the meaning within thepresent disclosure. The patent and scientific literature referred toherein is hereby incorporated by reference. The terms and definitionsprovided herein control, if in conflict with terms and/or definitionsincorporated by reference.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context dictates otherwise.

The terms “approximately” and “about” mean to be nearly the same as areferenced number or value including an acceptable degree of error forthe quantity measured given the nature or precision of the measurements.As used herein, the terms “approximately” and “about” should begenerally understood to encompass ±20% of a specified amount, frequencyor value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that term “about” or “approximately” can beinferred when not expressly stated.

The terms “administer,” “administration,” or “administering” as usedherein refer to (1) providing, giving, dosing and/or prescribing byeither a health practitioner or his authorized agent or under hisdirection a composition according to the disclosure, and (2) puttinginto, taking or consuming by the patient or person himself or herself, acomposition according to the disclosure. As used herein,“administration” of a composition includes any route of administration,including oral, intravenous, subcutaneous, intraperitoneal, andintramuscular.

As used herein, the term “subject” means a mammal, i.e., a human or anon-human mammal.

The phrase “a therapeutically effective amount” refers to an amount of atherapeutic agent that results in a measurable therapeutic response. Atherapeutic response may be any response that a user (e.g., a clinician)will recognize as an effective response to the therapy, includingimprovement of symptoms and surrogate clinical markers. Thus, atherapeutic response will generally be an amelioration or inhibition ofone or more symptoms of a disease or condition, e.g., stroke. Ameasurable therapeutic response also includes a finding that a symptomor disease is prevented or has a delayed onset, or is otherwiseattenuated by the therapeutic agent. thus, a “therapeutically effectiveamount” as used herein refers to an amount sufficient to reduce one ormore symptom(s) or condition(s) associated with an ischemic strokeincluding but not limited to hemorrhagic transformation, disruption ofthe blood-brain barrier, increase in hemoglobin levels, and mortality.

As used herein, “protein kinase C activator” or “PKC activator” means asubstance that increases the rate of the reaction catalyzed by proteinkinase C by binding to the protein kinase C.

As used herein “macrocyclic lactone” refers to a compound comprising amacrolide ring, i.e., a large macrocyclic lactone ring to which one ormore deoxy sugars may be attached.

Fatty acids according to the present disclosure may be saturated orunsaturated, branched or unbranched, and naturally-occurring orsynthetic.

The term “monounsaturated fatty acid” (MUFA) refers to a fatty acidcomprising a single C═C double bond with the remaining carbon atoms inthe chain singly-bonded; MUFAs are also called “monoenoic fatty acids.”Examples of MUFAs include, but are not limited to, oleic acid,myristoleic, acid and palm itoleic acid.

The term “cis-MUFA” refers to MUFAs wherein the hydrogen atoms adjacentto the C═C double bond are on the same side of the double bond.

The term “polyunsaturated fatty acid” (PUFA) refers to a fatty acidcomprising more than one C═C double bond; PUFAs are also called“polyenoic fatty acids.” PUFAs include, but are not limited to, omega-3fatty acids, omega-6 fatty acids, and omega-9 fatty acids; wherein thefirst C═C double bond is located 3, 6, and 9 carbons, respectively, fromthe last carbon in the chain farthest from the carboxylic acid group(known as the “omega carbon”). The abbreviation X:Y indicates an acylgroup containing X carbon atoms and Y double bonds. For example,linoleic acid would be abbreviated 18:2. Examples of PUFAs include, butare not limited to, linoleic acid (9,12-octadecadienoic acid);γ-linolenic acid (GLA; 6,9,12-octadecatrienoic acid); α-linolenic acid(9,12,15-octadecatrienoic acid); arachidonic acid(5,8,11,14-eicosatetraenoic acid); eicosapentanoic acid (EPA;5,8,11,14,17-eicosapentanoic acid); docosapentaenoic acid (DPA;7,10,13,16,19-docosapentaenoic acid); docosahexaenoic acid (DHA;4,7,10,13,16,19-docosahexanoic acid); and stearidonic acid(6,9,12,15-octadecatetraenoic acid). Sources of PUFAs include marinefish and vegetable oils derived from oil seed crops. PUFAs incommercially-developed plant oils may comprise, for example, linoleicacid and/or linolenic acid.

The term “cis-PUFA” refers to a PUFA wherein the carbon atoms adjacentto a C═C double bond are on the same side of the double bond.

The term “methylene-interrupted polyene” refers to a PUFA comprising twoor more cis C═C double bonds separated from each other by a singlemethylene (—CH₂—) group. The terms “non-methylene-interrupted polyene”and “polymethylene-interrupted fatty acid” refer to a PUFA having two ormore cis C═C double bonds separated by more than one methylene group.

Conjugated fatty acids such as conjugated linoleic acid(9-cis,11-trans-octadecadienoic acid, an isomer ofall-cis-9,12-octadecadienoic acid) have a conjugated diene, i.e., C═Cdouble bonds on adjacent carbons. Some evidence suggests that conjugatedlinoleic acid may have antitumor activity.

The term “cyclopropyl group” refers to a cycloalkane group of threecarbon atoms linked to form a three-membered ring (—CHCH₂CH—).

The term “epoxyl group” refers to a heterocyclic group comprising twocarbon atoms and an oxygen atom linked to form a three-membered ring(—CHOCH—).

The term “PUFA derivative” refers to a PUFA, or alcohol or esterthereof, in which at least one of the C═C double bonds iscyclopropanated or epoxidized.

The term “MUFA derivative” refers to a MUFA, or alcohol or esterthereof, in which the C═C double bond is cyclopropanated or epoxidized.

As used herein, “selective activation” means activation of one PKCisozyme, e.g., PKCε, to a greater detectable extent than any other PKCisozyme.

The term “neurodegeneration” refers to the progressive loss of structureor function of neurons, including death of neurons.

The term “pharmaceutically acceptable” refers to molecular entities andcompositions that are physiologically tolerable and do not typicallyproduce untoward reactions when administered to a subject.

While the present disclosure generally describes use of rTPA, otheranticoagulants and anticoagulant therapies suitable for the treatment ofstroke are also contemplated. Further, it is understood that the presentdisclosure is not limited to a specific manufactured type of TPA (e.g.,rTPA), but includes TPA generally.

The present disclosure generally relates to methods of treating strokecomprising an initial treatment of administering an anticoagulant, e.g.,rTPA, and a PKC activator followed by a subsequent treatment ofadministering a PKC activator. In some embodiments, the initialadministration of a PKC activator may extend the time that rTPA can beadministered after a stroke (e.g., after an ischemic event) while stillretaining efficacy. The subsequent administration of a PKC activator mayprovide additional protective, preventative, and/or regenerativebenefits such as, for example, antiapoptosis, antisynaptic loss, and/orsynaptogenesis. The methods disclosed herein may, for example, reducemortality, reduce hemorrhagic transformation, reduce disruptions to theblood-brain barrier (BBB), and/or reduce the level of assayedhemoglobin, wherein elevated hemoglobin is a risk factor for reducedreperfusion, greater infarction size, and/or mortality due to stroke.Further, the methods disclosed herein may improve cognitive capacity,learning, and/or memory following stroke, and may reverse stroke-inducedbrain injury and/or stroke-induced memory impairment.

Sliding Temporal Window

In the methods presently disclosed, a PKC activator may be administeredbefore, after, and/or at the same time as rTPA for the initialtreatment. In some embodiments of the present disclosure, rTPA and a PKCactivator are administered at the same time. Thus, the presentdisclosure contemplates “sliding temporal windows” for administration ofa PKC activator and rTPA to a subject. The term “sliding temporalwindow” refers to the notion that a PKC activator and rTPA can beadministered in any order to a subject that has suffered a stroke, atany time relative to one another, and at any time relative to when thestroke occurred.

At least four scenarios are contemplated:

Scenario 1:

In some embodiments of the present disclosure, a PKC activator may beadministered to a subject within a given time period after suffering astroke, followed by rTPA after another period of time. The PKC activatormay be administered at any time after the occurrence of a stroke,generally within about 24 hours. For example, the PKC activator may beadministered to a subject about 1 hour, about 2 hours, about 3 hours,about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours,about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,about 22 hours, about 23 hours, or about 24 hours after a stroke. TherTPA may then be administered to the subject about 1 hour, about 2hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, or about 24 hours afterthe PKC activator.

For example, in some embodiments, the PKC activator is administeredwithin 24 hours after the ischemic event, such as from about 1 hour toabout 12 hours or from about 2 hours to about 6 hours after the ischemicevent. rTPA is then administered within 24 hours after administration ofthe PKC activator, such as from about 1 hour to about 12 hours or fromabout 2 hours to about 6 hours after administration of the PKCactivator. In one embodiment, the PKC activator is administered withinabout 6 hours after the ischemic event and the rTPA is administeredwithin about 2 hours after administration of the PKC activator. Inanother embodiment, the PKC activator is administered about 3 hoursafter the ischemic event and the rTPA is administered about 2 hoursafter the PKC activator.

Scenario 2:

In some embodiments, rTPA may be administered to a subject within agiven time period after suffering a stroke, followed by administrationof a PKC activator after another period of time. The rTPA may beadministered at any time after the occurrence of a stroke, generallywithin about 24 hours. For example, the rTPA may be administered to asubject about 1 hour, about 2 hours, about 3 hours, about 4 hours, about5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours,about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours,about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23hours, or about 24 hours after a stroke. The PKC activator may then beadministered to the subject about 1 hour, about 2 hours, about 3 hours,about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours,about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,about 22 hours, about 23 hours, or about 24 hours after administrationof the rTPA.

For example, in some embodiments, the rTPA is administered within 24hours after the ischemic event, such as from about 1 hour to about 12hours or from about 2 hours to about 6 hours after the ischemic event.The PKC activator is then administered within 24 hours afteradministration of the rTPA, such as from about 1 hour to about 12 hoursor from about 2 hours to about 6 hours after the rTPA. In oneembodiment, rTPA is administered within about 6 hours after the ischemicevent and the PKC activator is administered within about 2 hours afterthe rTPA. In another embodiment, rTPA is administered about 3 hoursafter the ischemic event and the PKC activator is administered about 2hours after the rTPA.

Scenario 3:

In other embodiments of the present disclosure, a PKC activator may beadministered to a subject within a given time period after suffering astroke, followed by rTPA one or more times after another period of time,and further followed by administration of a PKC activator one or moretimes a period of time later. For example, the PKC activator may beadministered to a subject about 1 hour, about 2 hours, about 3 hours,about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours,about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,about 22 hours, about 23 hours, or about 24 hours after a stroke. TherTPA may then be administered to the subject about 1 hour, about 2hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, or about 24 hours afteradministration of the PKC activator. Thereafter, another PKC activatormay be administered about 1 hour, about 2 hours, about 3 hours, about 4hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours,about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours,about 23 hours, or about 24 hours after administration of the rTPA. ThePKC activator administered before and after rTPA may be the same ordifferent.

Similarly, rTPA may be administered to a subject one or more timeswithin a given time period after having suffered a stroke, followed by aPKC activator one or more times after another time period, and furtherfollowed by administration of the same or a different PKC activator oneor more times a period of time later.

Scenario 4:

In yet other embodiments, a PKC activator and rTPA may be administeredat the same time to a subject after suffering a stroke. This may be doneby directly administering a composition comprising a PKC activator andrTPA, or administering a composition comprising a PKC activator and aseparate composition comprising rTPA in rapid succession, one after theother in either order (i.e., the composition comprising a PKC activatormay be administered first or the composition comprising rTPA may beadministered first).

In some embodiments, the present disclosure provides a method forextending the therapeutic window for treating ischemic stroke with rTPAcomprising administering a PKC activator before, after, or at the sametime as rTPA. The recommended time period for administering rTPA (e.g.,Activase®) is about 3 hours. In one embodiment of the presentdisclosure, for example, a PKC activator is administered to a subjectabout 2 hours after a stroke followed by administration of rTPA about 6hours later (i.e., about 8 hours after the stroke). In anotherembodiment, rTPA is administered to a subject about 6 hours after astroke followed by administration of a PKC activator about 2 hours later(i.e., about 8 hours after the stroke).

At least one embodiment of the present disclosure provides for treatmentof a subject who has suffered a stroke before it is known whether thesubject suffered an ischemic stroke or a hemorrhagic stroke. Forexample, the present disclosure provides for a method of identifying asubject who has suffered a stroke, administering atherapeutically-effective amount of a PKC activator, and determiningwhether the subject suffered an ischemic stroke or a hemorrhagic stroke.The determination regarding the type of stroke suffered may be made byany suitable means known in the medical arts including, for example, acomputed tomography (CT) scan. If the subject suffered an ischemicstroke, a therapeutically-effective amount of rTPA may be administered.If the subject suffered a hemorrhagic stroke, however, rTPA is notadministered. Thus, in some embodiments of the present disclosure,extending the therapeutic time window for treating stroke with rTPAallows for a determination of whether a subject suffered an ischemicstroke or a hemorrhagic stroke.

In the methods disclosed herein the initial treatment of rTPA and PKCactivator, for example described in scenarios 1-4, is followed by asubsequent treatment of a PKC activator. The subsequent treatment of PKCactivator may be initiated, for example, from about 10 hours to about 32hours after the ischemic event, such as about 24 hours after theischemic event. The PKC activators administered in the initial treatmentand the subsequent treatment may be the same or different. In someembodiments, the PKC activator is administered from 1-3 times per week.In some embodiments, the duration of treatment ranges from about 1 weekto about 10 weeks, such as from about 1 week to about 6 weeks, forexample about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about5 weeks, or about 6 weeks.

PKC Activators

In some embodiments of the present disclosure, the PKC activator mayactivate PKCε at least 1-fold, 2-fold or 5-fold over other PKC isozymes,for example as measured via a PKC activation assay as described herein.Upon activation, PKC enzymes are translocated to the plasma membrane byRACK (receptor for activated C-kinase) proteins, which aremembrane-bound receptors for activated PKC. In general, upon activation,PKC enzymes are translocated to the plasma membrane by RACK proteins.Other indicia of PKC activation include phosphorylation at specificC-terminal serine/threonine residues byphosphatidylinositol-trisphosphate-dependent kinase (PDK1), with atleast two additional phosphorylations and/or autophosphorylations ofwell-conserved sequences in each enzyme of the PKC family. Activation ofPKC is described, for example, in Sun et al., Recent Patents CNS DrugDiscov. (2006), vol. 1, pp. 147-56.

PKC activators suitable for the methods, compositions, and kitsdisclosed herein include, for example, macrocyclic lactones, e.g.,bryostatin and neristatin classes, that act to stimulate PKC. Of thebryostatin class of compounds, bryostatin-1 has been shown to activatePKC without tumor promotion. Bryostatin-1 may be particularly useful asa PKC activator because the dose response curve is biphasic andbryostatin-1 demonstrates differential regulation of PKC isozymesincluding PKCα, PKCδ and PKCε. Bryostatin-1 has undergone toxicity andsafety studies in animals and humans, and is actively investigated as ananti-cancer agent.

Macrocyclic lactones generally comprise 14-, 15-, or 16-membered lactonerings. Macrolides belong to the polyketide class of natural products.Macrocyclic lactones and derivatives thereof are described, for example,in U.S. Pat. Nos. 6,187,568; 6,043,270; 5,393,897; 5,072,004; 5,196,447;4,833,257; and 4,611,066; and 4,560,774; each incorporated by referenceherein in its entirety. Those patents describe various compounds andvarious uses for macrocyclic lactones including their use as ananti-inflammatory or anti-tumor agent. Szallasi et al. J. Biol. Chem.(1994), vol. 269, pp. 2118-2124; Zhang et al., Cancer Res. (1996), vol.56, pp. 802-808; Hennings et al. Carcinogenesis (1987), vol. 8, pp.1343-1346; Varterasian et al. Clin. Cancer Res. (2000), vol. 6, pp.825-828; Mutter et al. Bioorganic & Med. Chem. (2000), vol. 8, pp.1841-1860; each incorporated by reference herein in its entirety. Thebryostatin and neristatin compounds were originally isolated from themarine bryozoan Bugula neritina L.

In one embodiment, for example, the PKC activator is a macrocycliclactone, such as a bryostatin or neristatin. Bryostatins include, forexample, bryostatin-1, bryostatin-2, bryostatin-3, bryostatin-4,bryostatin-5, bryostatin-6, bryostatin-7, bryostatin-8, bryostatin-9,bryostatin-10, bryostatin-11, bryostatin-12, bryostatin-13,bryostatin-14, bryostatin-15, bryostatin-16, bryostatin-17, andbryostatin-18. In at least one embodiment, the bryostatin isbryostatin-1. Neristatins suitable for the present disclosure include,for example, neristatin-1.

Analogs of bryostatin, commonly referred to as bryologs, are oneparticular class of PKC activators that are suitable for use in thepresent disclosure. Table 1 summarizes structural characteristics ofseveral bryologs and demonstrates variability in their affinity for PKC(ranging from 0.25 nM to 10 μM). Structurally, they are all similar.While bryostatin-1 has two pyran rings and one 6-membered cyclic acetal,in most bryologs one of the pyrans of bryostatin-1 is replaced with asecond 6-membered acetal ring. This modification reduces the stabilityof bryologs, relative to bryostatin-1, for example, in both strong acidor base, but has little significance at physiological pH. Bryologs alsohave a lower molecular weight (ranging from about 600 g/mol to 755g/mol), as compared to bryostatin-1 (988), a property which facilitatestransport across the blood-brain barrier.

TABLE 1 Bryologs. PKC Affin Name (nM) MW Description Bryo- 1.35 988 2pyran + 1 cyclic acetal + macrocycle statin-1 Analog 1 0.25 737 1pyran + 2 cyclic acetal + macrocycle Analog 2 6.50 723 1 pyran + 2cyclic acetal + macrocycle Analog 7a — 642 1 pyran + 2 cyclic acetals +macrocycle Analog 7b 297 711 1 pyran + 2 cyclic acetals + macrocycleAnalog 7c 3.4 726 1 pyran + 2 cyclic acetals + macrocycle Analog 7d10000 745 1 pyran + 2 cyclic acetals + macrocycle, acetylated Analog 88.3 754 2 cyclic acetals + macrocycle Analog 9 10000 599 2 cyclicacetals

Analog 1 exhibits the highest affinity for PKC. Wender et al., Curr.Drug Discov. Technol. (2004), vol. 1, pp. 1-11; Wender et al. Proc.Natl. Acad. Sci. (1998), vol. 95, pp. 6624-6629; Wender et al., J. Am.Chem. Soc. (2002), vol. 124, pp. 13648-13649, each incorporated byreference herein in their entireties. Only Analog 1 exhibits a higheraffinity for PKC than bryostatin. Analog 2, which lacks the A ring ofbryostatin-1, is the simplest analog that maintains high affinity forPKC. In addition to the active bryologs, Analog 7d, which is acetylatedat position 26, has virtually no affinity for PKC.

B-ring bryologs may also be used in the present disclosure. Thesesynthetic bryologs have affinities in the low nanomolar range. Wender etal., Org Lett. (2006), vol. 8, pp. 5299-5302, incorporated by referenceherein in its entirety. B-ring bryologs have the advantage of beingcompletely synthetic, and do not require purification from a naturalsource.

A third class of suitable bryostatin analogs is the A-ring bryologs.These bryologs have slightly lower affinity for PKC than bryostatin-1(6.5, nM, 2.3 nM, and 1.9 nM for bryologs 3, 4, and 5, respectively) anda lower molecular weight.

Bryostatin analogs are described in U.S. Pat. Nos. 6,624,189 and7,256,286.

A number of derivatives of diacylglycerol (DAG) bind to and activatePKC. Niedel et al., Proc. Natl. Acad. Sci. (1983), vol. 80, pp. 36-40;Mori et al., J. Biochem. (1982), vol. 91, pp. 427-431; Kaibuchi et al.,J. Biol. Chem. (1983), vol. 258, pp. 6701-6704. However, DAG and DAGderivatives are of limited value as drugs. Activation of PKC bydiacylglycerols is transient, because they are rapidly metabolized bydiacylglycerol kinase and lipase. Bishop et al. J. Biol. Chem. (1986),vol. 261, pp. 6993-7000; Chuang et al. Am. J. Physiol. (1993), vol. 265,pp. C927-C933; incorporated by reference herein in their entireties. Thefatty acid substitution determines the strength of activation.Diacylglycerols having an unsaturated fatty acid are most active. Thestereoisomeric configuration is important; fatty acids with a 1,2-snconfiguration are active while 2,3-sn-diacylglycerols and1,3-diacylglycerols do not bind to PKC. Cis-unsaturated fatty acids maybe synergistic with diacylglycerols. In at least one embodiment, theterm “PKC activator” expressly excludes DAG or DAG derivatives.

Isoprenoids are PKC activators also suitable for the present disclosure.Farnesyl thiotriazole, for example, is a synthetic isoprenoid thatactivates PKC with a K_(d) of 2.5 Farnesyl thiotriazole, for example, isequipotent with dioleoylglycerol, but does not possess hydrolyzableesters of fatty acids. Gilbert et al., Biochemistry (1995), vol. 34, pp.3916-3920; incorporated by reference herein in its entirety. Farnesylthiotriazole and related compounds represent a stable, persistent PKCactivator. Because of its low molecular weight (305.5 g/mol) and absenceof charged groups, farnesyl thiotriazole would be expected to readilycross the blood-brain barrier.

Octylindolactam V is a non-phorbol protein kinase C activator related toteleocidin. The advantages of octylindolactam V (specifically the(−)-enantiomer) include greater metabolic stability, high potency(EC₅₀=29 nM) and low molecular weight that facilitates transport acrossthe blood brain barrier. Fujiki et al. Adv. Cancer Res. (1987), vol. 49pp. 223-264; Collins et al. Biochem. Biophys. Res. Commun. (1982), vol.104, pp. 1159-4166, each incorporated by reference herein in itsentirety.

Gnidimacrin is a daphnane-type diterpene that displays potent antitumoractivity at concentrations of 0.1 nM-1 nM against murine leukemias andsolid tumors. It acts as a PKC activator at a concentration of 0.3 nM inK562 cells, and regulates cell cycle progression at the G1/S phasethrough the suppression of Cdc25A and subsequent inhibition of cyclindependent kinase 2 (Cdk2) (100% inhibition achieved at 5 ng/ml).Gnidimacrin is a heterocyclic natural product similar to bryostatin, butsomewhat smaller (MW=774.9 g/mol).

Iripallidal is a bicyclic triterpenoid isolated from Iris pallida.Iripallidal displays anti-proliferative activity in a NCI 60 cell linescreen with GI₅₀ (concentration required to inhibit growth by 50%)values from micromolar to nanomolar range. It binds to PKCα with highaffinity (K₁=75.6 nM). It induces phosphorylation of Erk1/2 in aRasGRP3-dependent manner. Its molecular weight is 486.7 g/mol.Iripallidal is about half the size of bryostatin and lacks chargedgroups.

Ingenol is a diterpenoid related to phorbol but less toxic. It isderived from the milkweed plant Euphorbia peplus. Ingenol3,20-dibenzoate, for example, competes with [3H] phorbol dibutyrate forbinding to PKC (K₁=240 nM). Winkler et al., J. Org. Chem. (1995), vol.60, pp. 1381-1390, incorporated by reference herein. Ingenol-3-angelateexhibits antitumor activity against squamous cell carcinoma and melanomawhen used topically. Ogbourne et al. Anticancer Drugs (2007), vol. 18,pp. 357-362, incorporated by reference herein.

Napthalenesulfonam ides, includingN-(n-heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) andN-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, are members ofanother class of PKC activators. SC-10 activates PKC in acalcium-dependent manner, using a mechanism similar to that ofphosphatidylserine. Ito et al., Biochemistry (1986), vol. 25, pp.4179-4184, incorporated by reference herein. Naphthalenesulfonamides actby a different mechanism than bryostatin and may show a synergisticeffect with bryostatin or member of another class of PKC activators.Structurally, naphthalenesulfonamides are similar to the calmodulin(CaM) antagonist W-7, but are reported to have no effect on CaM kinase.

Diacylglycerol kinase inhibitors may also be suitable as PKC activatorsin the present disclosure by indirectly activating PKC. Examples ofdiacylglycerol kinase inhibitors include, but are not limited to,6-(2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)ethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one(R59022) and[3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quinazolinone(R59949).

A variety of growth factors, such as fibroblast growth factor 18(FGF-18) and insulin growth factor, function through the PKC pathway.FGF-18 expression is up-regulated in learning, and receptors for insulingrowth factor have been implicated in learning. Activation of the PKCsignaling pathway by these or other growth factors offers an additionalpotential means of activating PKC.

Growth factor activators, including 4-methyl catechol derivatives like4-methylcatechol acetic acid (MCBA) that stimulate the synthesis and/oractivation of growth factors such as NGF and BDNF, also activate PKC aswell as convergent pathways responsible for synaptogenesis and/orneuritic branching.

The PKC activators according to the present disclosure include fattyacids such as unsaturated fatty acids, e.g., MUFAs and/or PUFAs, andderivatives thereof in which at least one C═C double bond is replaced bya cyclopropyl group (i.e., “cyclopropanated” double bond) or an epoxylgroup (i.e., “epoxidized” double bond). In some embodiments, all of theC═C double bonds of an unsaturated fatty acid are replaced bycyclopropyl groups and/or epoxyl groups. In some embodiments, the fattyacid derivatives may comprise both cyclopropyl groups and epoxyl groups.

In some embodiments of the present disclosure, the PKC activatorcomprises a fatty acid derivative to treat stroke. In some embodiments,for example, the fatty acid derivatives such as PUFA and/or MUFAderivatives may activate PKCε at low (e.g., nanomolar) concentrations.

The terminal functional group of the fatty acid derivatives may be, forexample, a free carboxylic acid (—CO₂), an alcohol (—CHOH), or an ester(—CO₂R) such as a monoester or polyester. The alkyl group (R) of theester may be straight or branched including, for example, methyl, ethyl,propyl, isopropyl, butyl, isobutyl, secbutyl, tert-butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, and tetradecylgroups. An ester may also be formed from a fatty acid linked to a fattyalcohol in an ester linkage. Other alkyl esters contemplated includealiphatic alcohol esters and aromatic alcohol esters. In one embodiment,for example, the alcohol ester is a propylene glycol ester. In anotherembodiment, the alcohol ester is a glycerol ester. Glycerol esters offatty acids include, for example, glycerol fatty acid ester, glycerolacetic acid fatty acid ester, glycerol lactic acid fatty acid ester,glycerol citric acid fatty acid ester, glycerol succinic acid fatty acidester, glycerol diacetyl tartaric acid fatty acid ester, glycerol aceticacid ester, polyglycerol fatty acid ester, and polyglycerol condensedricinoleic acid ester. Glycerol derivatives are biologically importantbecause fatty acids may be conjugated to glycerol in the form ofphosphatidylcholine, phosphatidylserine, and phosphatidic acids. Forexample, triacylglycerols (or triglycerides) are compounds in which thecarboxyl groups of three fatty acids are esterified to the hydroxyls ofall three carbons of glycerol. Esterifying the carboxylic acidfacilitates transport across the blood-brain barrier by eliminating thenegative charge; an alcohol group also facilitates transport across theblood-brain barrier.

MUFAs that can be the basis for the fatty acid derivatives of thepresent disclosure include, but are not limited to, fatty acids with thefollowing structure:

CH₃(CH₂)xCH═CH(CH₂)yCOOH

wherein each of x and y, independent of one another, is an odd integerfrom 3 to 11. Examples include cis- and trans-MUFAs such as oleic acid,elaidic acid, obtusilic acid, caproleic acid, lauroleic acid, lindericacid, myristoleic acid, palm itoleic acid, vaccenic acid, gadoleic acid,erucic acid, and petroselinic acid. Examples of MUFA alcohols include,for example, elaidic alcohol, oleyl alcohol, and 1-monolinoleylrac-glycerol. Specific examples of cyclopropanated and epoxidized MUFAderivatives include eliadic alcohol cyclopropane (BR-106), eliadic acidcyclopropane (BR-107), oleyl alcohol cyclopropane (BR-108), andepoxystearic acid (BR-116). See FIG. 5.

Naturally cyclopropanated or epoxidized MUFAS or ester or alcoholderivatives thereof contemplated for the methods presently disclosedinclude malvenic acid, vernolic acid, and sterculic acid. An exemplarycompound is vernolic acid methyl ester (BR-117).

PUFAs that can be the basis for fatty acid derivatives of the presentdisclosure include, but are not limited to, fatty acids with thefollowing structure:

CH₃(CH₂)₄(CH═CHCH₂)x(CH₂)yCOOH

wherein x and y are each independently integers ranging from 2 to 6,including methylene- and/or polymethylene-interrupted polyenes. Theseare omega-6 PUFAs. Examples include, but are not limited to, linoleicacid, γ-linoleic acid, arachidonic acid, and adrenic acid, which havethe following structures:

linoleic acid CH₃(CH₂)₄(CH═CHCH₂)₂(CH₂)₆COOH

γ-linolenic acid CH₃(CH₂)₄(CH═CHCH₂)₃(CH₂)₃COOH

arachidonic acid CH₃(CH₂)₄(CH═CHCH₂)₄(CH₂)₂COOH

adrenic acid CH₃(CH₂)₄(CH═CHCH₂)₄(CH₂)₄COOH

The linoleic acid derivative DCP-LA(2-[(2-pentylcyclopropyl)methyl]cyclopropaneoctanoic acid) (BR-101) isone of the few known isoform-specific activators of PKC known. See FIG.5. DCP-LA selectively activates PKCε with a maximal effect at 100 nM.(Kanno et al., J. Lipid Res. (2006) vol. 47, pp. 1146-1156. Like SC-10,DCP-LA interacts with the phosphatidylserine binding site of PKC,instead of the diacylglycerol binding site.

Further examples of PUFAs that can be the basis for fatty acidderivatives of the present disclosure include the following structure:

CH₃CH₂(CH═CHCH₂)x(CH₂)yCOOH

wherein x and y are each independently integers ranging from 2 to 6,including methylene- and/or polymethylene-interrupted polyenes. Theseare omega-3 PUFAs. Examples include, but are not limited to, α-linoleicacid, docosahexaenoic acid, eicosapentaenoic acid, and eicosatetraenoicacid, which have the following structures:

α-linolenic acid CH₃CH₂(CH═CHCH₂)₃(CH₂)₆COOH

eicosatetraenoic acid CH₃CH₂(CH═CHCH₂)₄(CH₂)₅COOH

eicosapentaenoic acid CH₃CH₂(CH═CHCH₂)₅(CH₂)₂COOH

docosahexaenoic acid CH₃CH₂(CH═CHCH₂)₆(CH₂)₂COOH

PUFA derivatives include PUFAs (carboxylic acid, alcohol, or esterterminal groups) wherein at least one of the C═C double bonds iscyclopropanated or epoxidized. Examples of cis-PUFA esters include thefollowing structures:

CH₃(CH₂)₄(CH═CHCH₂)x(CH₂)yCOOR

CH₃CH₂(CH═CHCH₂)x(CH₂)yCOOR

where x and y are each independently integers ranging from 2 to 6, and Ris an alkyl group. In some embodiments, R is the alkyl group of analcohol such as a monohydric or polyhydric alcohol. Examples of alcoholsinclude, but are not limited to, methanol, ethanol, propanol, butanol,pentanol, glycerol, mannitol, and sorbitol. In such cases, the alcoholmay comprise a branched or unbranched alkyl chain or may comprise anaromatic alkyl such as a phenolic alcohol. Examples of PUFA derivativesinclude, but are not limited to, linoleic alcohol dicyclopropane(BR-105), linolenic alcohol tricyclopropane (BR-104), and vernolic acidmethyl ester cyclopropane (BR-109). See FIG. 5.

In some embodiments, the PUFA derivative is a PUFA or ester or alcoholthereof wherein at least one of the C═C double bonds has beencyclpropanated or epoxidized. In some embodiments, for example, the PUFAderivative comprises a PUFA or ester or alcohol thereof with from two tosix cyclopropanated or epoxidized double bonds. In at least oneembodiment, the PUFA derivative comprises a PUFA or alcohol or esterthereof with three cyclopropanated or epoxidized double bonds. The PUFAderivatives of the present disclosure may also comprise both cyclopropylgroups and epoxyl groups.

In some embodiments, the PUFA derivative may comprise an epoxidizedcis-PUFA alcohol such as linoleic alcohol dicyclopropane or linolenicalcohol tricyclopropane.

PUFAs that may form the basis of the cyclopropanated and/or epoxidizedfatty acids according to the present disclosure include, but are notlimited to, arachidonic acid (AA), docosahexaenoic acid (DHA), andeicosapentaenoic acid (EPA). Exemplary PUFA derivatives includedocahexaenonic acid methyl ester hexacyclopropane (BR-111);eicosapentaenoic acid methyl ester pentacyclopropane (BR-114); andarachidonic acid methyl ester tetracyclopropane (BR-115). See FIG. 5.

In one embodiment, the PKC activator comprises a cyclopropanated PUFAderivative of DHA with the following structure:

wherein R is H or an alkyl group. In one embodiment, R is methyl (BR-111or DHA-CB6 methyl ester), ormethyl-3-(2-((2-((2-((2-((2-((2-ethylcyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)methyl)-cyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)propanoate.

In another embodiment, the PKC activator comprises a PUFA derivativewith the following structure:

This compound is BR-114 (EPA-CP5 or methyl4-(2((2-((2-((2-ethylcyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)methyl)-cyclopropyl)butanoatemethyl ester).

In still another embodiment, the PKC activator comprises a PUFAderivative with the following structure:

This compound is BR-115 (AA-CP4 or methyl4-(2-((2-((2-((-pentylcyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)butanoatemethyl ester).

In another embodiment, the PKC activator comprises a PUFA derivativewith the following structure:

wherein R is H or an alkyl ester. In one embodiment, R is methyl.

Methods of Synthesis

Fatty acids, and esters and alcohols thereof, can be obtained or madefrom purification from natural sources, e.g., fish oil, flaxseed oil,soybeans, rapeseed oil, or algae, or synthesized using a combination ofmicrobial enzymatic synthesis and chemical synthesis. As one example,fatty acid methyl esters can be produced by the transesterification oftriglycerides of refined/edible type oils using methanol and anhomogeneous alkaline catalyst.

Methods of cyclopropanation of double bonds in hydrocarbons are known inthe art. For example, the modified Simmons-Smith reaction is a standardmethod for converting double bonds to cyclopropanes. Tanaka andNishizaki, Bioorg. Med. Chem. Lett. (2003), vol. 13, pp. 1037-1040;Kawabata and Nishimura, J. Tetrahedron (1967), vol. 24, pp. 53-58;Denmark and Edwards, J. Org. Chem. (1991), vol. 56, pp. 6974-6981. Inthis reaction, treatment of alkenes with metal carbenoids, e.g.,methylene iodide and diethylzinc, result in cyclopropanation of thealkene. See also Ito et al., Organic Syntheses (1988), vol. 6, p. 327.Cyclopropanation of methyl esters of was also effected usingdiazomethane in the presence of palladium (II) acetate as catalyst.Gangadhar et al., J. Am. Oil Chem. Soc. (1988), vol. 65, pp. 601-606.

Methods of epoxidation are also known in the art and typically involvereaction of fatty acid dioxiranes in organic solvents. Sonnet et al., J.Am. Oil Chem. Soc. (1995), vol. 72, pp. 199-204. As one example,epoxidation of PUFA double bonds can be achieved using dimethyldioxirane(OMD) as the epoxidizing agent. Grabovskiy et al., Helvetica ChimicaActa (2006) vol. 89, pp. 2243-22453.

The present disclosure contemplates treatment of neurological injuriesand/or diseases associated with stroke. Without being limited to anyparticular mechanism, selective activation of PKCε may result inincreased activation of alpha-secretase, e.g., tumor necrosisfactor-α-converting enzyme (TACE), with a concomitant decrease inproduction of Aβ. However, this appears to occur mainly in non-neuronalcells such as fibroblasts. Activation of PKCε may also inducesynaptogenesis or prevent apoptosis following stroke or in Alzheimer'sdisease. Activation of PKCε may also protect neurons from Aβ-mediatedneurotoxicity through inhibition of GSK-3β.

The methods disclosed herein may reduce mortality 24 hours after stroke.For example, mortality after 24 hours may be reduced by at least 20%, atleast 30%, at least 40%, or at least 50%. In at least one embodiment,the initial administration of a PKC activator and rTPA followed bysubsequent administration of a PKC activator reduces mortality 24 hoursafter stroke by at least 40%.

In some embodiments, the methods of treatment disclosed herein mayreduce disruption of the blood-brain barrier after stroke and/or mayreduce hemorrhagic transformation. In some embodiments, for example,administering a PKC activator and rTPA after a stroke followed bysubsequent administration of a PKC activator may reduce hemoglobinlevels, wherein a reduction in hemoglobin indicates a reduction inhemorrhagic transformation and/or a reduction in disruption of theblood-brain barrier. In some embodiments, the hemoglovin level isreduced by about 30%, about 35%, about 40%, about 45%, about 50%, about55%, or about 60%. In at least one embodiment, for example, thehemoglobin level is reduced by about 50%. Reduced disruption of theblood-brain barrier may also be assessed by measuring extravasation ofalbumin. DiNapoli et al., Neurobiology of Aging (2008), vol. 29, pp.753-764.

Further, in some embodiments of the present disclosure, the size of theinfarction due to stroke (e.g., tissue damage caused by an ischemicevent) may be limited and/or reduced.

Formulation and Administration

The formulations of the pharmaceutical compositions described herein maybe prepared by any suitable method known in the art of pharmacology. Ingeneral, such preparatory methods include bringing the active ingredientinto association with a carrier or one or more other accessoryingredients, then, if necessary or desirable, shaping or packaging theproduct into a desired single- or multi-dose unit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions suitable forethical administration to humans, it will be understood by skilledartisan that such compositions are generally suitable for administrationto animals of all sorts. Modification of pharmaceutical compositionssuitable for administration to humans order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and performsuch modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions ofthe invention is contemplated include, but are not limited to, humansand other primates, and other mammals.

In some embodiments, the PKC activator and anticoagulant, e.g., rTPA,are formulated together. In other embodiments, the PKC activator andrTPA are formulated separately.

The compositions disclosed herein may be administrated by any suitableroute including oral, parenteral, transmucosal, intranasal, inhalation.or transdermal routes. Parenteral routes include intravenous,intra-arteriolar, intramuscular, intradermal, subcutaneous,intraperitoneal, intraventricular, intrathecal, and intracranialadministration. A suitable route of administration may be chosen topermit crossing the blood-brain barrier. Rapoport et al., J. Lipid Res.(2001) vol. 42, pp. 678-685.

The compositions disclosed herein may be formulated according toconventional methods, and may include any pharmaceutically acceptableadditives, such as excipients, lubricants, diluents, flavorants,colorants, buffers, and disintegrants. See e.g., Remington'sPharmaceutical Sciences, 20^(th) Ed., Mack Publishing Co. 2000.

In some embodiments, the PKC activator is formulated in a solid oraldosage form. For oral administration, the composition may take the formof a tablet or capsule prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods generally known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-phydroxybenzoates orsorbic acid). The preparations may also comprise buffer salts,flavoring, coloring and sweetening agents as appropriate.

In other embodiments of the present disclosure, the PKC activator may beformulated for parenteral administration such as bolus injection orcontinuous infusion. Formulations for injection may be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The compositions may take such forms as suspensions,solutions, dispersions, or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents.

In some embodiments, the PKC activator may be formulated with apharmaceutically-acceptable carrier for administration. Pharmaceuticallyacceptable carriers include, but are not limited to, one or more of thefollowing: excipients; surface active agents; dispersing agents; inertdiluents; granulating and disintegrating agents; binding agents;lubricating agents; sweetening agents; flavoring agents; coloringagents; preservatives; physiologically degradable compositions such asgelatin; aqueous vehicles and solvents; oily vehicles and solvents;suspending agents; dispersing or wetting agents; emulsifying agents,demulcents; buffers; salts; thickening agents; fillers; emulsifyingagents; antioxidants; antibiotics; antifungal agents; stabilizingagents; and pharmaceutically acceptable polymeric or hydrophobicmaterials. Other “additional ingredients” which may be included in thepharmaceutical compositions of the invention are generally known in theart and may be described, for example, in Remington's PharmaceuticalSciences, Genaro, ed., Mack Publishing Co., Easton, Pa., 1985,incorporated by reference herein.

In some embodiments, the PKC activator may be formulated with ahydrophobic carrier for administration. Hydrophobic carriers includeinclusion complexes, dispersions (such as micelles, microemulsions, andemulsions), and liposomes. Exemplary hydrophobic carriers includeinclusion complexes, micelles, and liposomes. See, e.g., Remington's:The Science and Practice of Pharmacy 20th ed., ed. Gennaro, Lippincott:Philadelphia, Pa. 2003. The PKC activators presently disclosed may beincorporated into hydrophobic carriers, for example as at least 1%, atleast 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, or at least 90% ofthe total carrier by weight. In addition, other compounds may beincluded either in the hydrophobic carrier or the solution, e.g., tostabilize the formulation.

In some embodiments, the PKC activator may also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the PKC activator may beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

In another embodiment, the PKC activator may be delivered in a vesicle,such as a micelle, liposome, or an artificial low-density lipoprotein(LDL) particle. See, e.g., U.S. Pat. No. 7,682,627.

The doses for administration may suitably be prepared so as to deliverfrom about 1 mg to about 10 g, such as from about 10 mg to about 1 g, orfor example, from about 250 mg to about 500 mg of the PKC activator perday. When prepared for topical administration or parenteral formulationsthey may be made in formulae containing from about 0.01% to about 60% byweight of the final formulation, such as from about 0.1% to about 30% byweight, such as from about 1% to about 10% by weight. A suitable dosecan be determined by methods known in the art and according toclinically relevant factors such as the age of the patient.

In at least one embodiment, the PKC activator is formulated forintravenous administration. The PKC activator may be administered in adose ranging from about 5 μg/m² to about 50 μg/m², for example fromabout 10 μg/m² to about 30 μg/m², or from about 25 μg/m² to about 50μg/m². In some embodiments, for example, the initial administration of aPKC activator ranges from about 25 μg/m² to about 50 μg/m². In someembodiments, the subsequent administration of a PKC activator rangesfrom about 5 μg/m² to about 30 μg/m², for example about 10 μg/m², about15 μg/m², or about 20 μg/m². In some embodiments, the PKC activator andrTPA are both formulated for intravenous administration. The rTPA may beformulated for intravenous administration of a dose of about 0.9 mg/kg.The PKC and rTPA may be formulated together for intravenousadministration, or they may be formulated separately for intravenousadministration.

Kits

The present disclosure further relates to kits that may be utilized forpreparing and administering pharmaceutical compositions of ananticoagulant, e.g., rTPA, and a PKC activator disclosed herein to asubject in need thereof. The kits may also comprise devices such assyringes for administration of the pharmaceutical compositions describedherein.

In some embodiments, the kits may comprise one or more vials, syringes,needles, ampules, cartridges, bottles or other such vessels for storingand/or subsequently mixing compositions of rTPA and PKC activatordisclosed herein. In certain embodiments, the devices, syringes,ampules, cartridges, bottles or other such vessels for storing and/orsubsequently mixing the compositions of rTPA and a PKC activatordisclosed herein may, or may not have more than one chamber.

In still further embodiments, the compositions of rTPA and a PKCactivator disclosed herein may be stored in one or more graduatedvessels (such as a syringe or syringes or other device useful formeasuring volumes).

In certain embodiments, the kits may comprise pharmaceuticalcompositions of rTPA and a PKC activator stored within the same orseparate ampules, vials, syringes, cartridges, bottles or other suchvessels.

The kits may also comprise one or more anesthetics, preferably localanesthetics. In certain embodiments, the anesthetics are in aready-to-use formulation, such as, for example an injectable formulation(optionally in one or more pre-loaded syringes) or a formulation thatmay be applied topically to an area where the compositions of rTPA andPKC activator disclosed herein are to be administered.

Topical formulations of anesthetics may be in form an anesthetic appliedto a pad, swab, towelette, disposable napkin, cloth, patch, bandage,gauze, cotton ball, Q-tip™, ointment, cream, gel, paste, liquid, or anyother topically applied formulation. Anesthetics for use with thepresent invention may include, but are not limited to lidocaine,marcaine, cocaine and xylocaine, for example.

The kits may also contain instructions relating to the use of thepharmaceutical compositions of rTPA and a PKC activator and proceduresfor mixing, diluting or combining formulations of rTPA and a PKCactivator. The instructions may also contain directions for properlydiluting formulations of rTPA and/or a PKC activator to obtain a desiredpH or range of pHs and/or a desired specific activity and/or proteinconcentration after mixing but prior to administration. The instructionsmay also contain dosing information. The instructions may also containmaterial directed to methods for selecting subjects for treatment withthe disclosed pharmaceutical compositions of rTPA and a PKC activator.The kits may also include additional buffers, syringes, needles,needle-less injection devices, sterile pads or swabs.

In some embodiments of the present disclosure, the kit comprises acomposition comprising rTPA and one or more compositions comprising aPKC activator, for example at least two compositions each comprising aPKC activator. The two or more compositions may comprise the same ordifferent PKC activators, and may be formulated for the same dose ordifferent doses of PKC activator.

The following examples are intended to illustrate the present disclosurewithout, however, being limiting in nature. It is understood that theskilled artisan will envision additional embodiments consistent with thedisclosure provided herein.

EXAMPLES Example 1 Focal Ischemia Model of Stroke

A transient animal model of focal ischemia was used for theseexperiments. The middle cerebral artery (MCA) was surgically dissectedand occluded in anesthetized rats by ligature, followed by reperfusionafter a defined period (about 2 hours). Animal models transient ischemiavia occlusion of the MCA (MCAO) are well-known and described in, e.g.,Sicard and Fisher, Exp. & Transl. Stroke Med. (2009), vol. 1, pp. 1-7.

Example 2 Drug Administration

In a first experiment, rTPA was administered intravenously (˜0.9 mg/kg)6 hours after the ischemic event, followed 2 hours later with a singleintravenous administration of bryostatin-1 in a dosage range of fromabout 25 μg/m² to 50 μg/m².

In a second experiment, bryostatin-1 was administered intravenously(about 25 μg/m² to 50 μg/m²) 2 hours after the ischemic event, followedby intravenous administration of rTPA (˜0.9 mg/kg) about 6 hours later.

In a third experiment, rTPA was administered intravenously (˜0.9 mg/kg)2 hours after the ischemic event, followed by intravenous administrationof bryostatin-1 in a dosage range of from about 25 μg/m² to 50 μg/m²about 6 hours later.

Example 3 Results

1. Mortality.

rTPA given 6 hours after the stroke, followed 2 hours later withbryostatin-1 led to 0% mortality 24 hours later (N=9 animals). Incontrast, if rTPA was given 6 hours after the stroke, in the absence ofsubsequent treatment with bryostatin, 44% mortality was observed (N=6animals).

2. Hemorrhage, Edema, and Blood-Brain Barrier Disruptions.

Bryostatin-1 administered 2 hours after the stroke, followed 6 hourslater by rTPA, resulted in a 50% reduction of assayed hemoglobin in thecortex and striatum, as compared to rTPA given 6 hours after the strokewithout prior bryostatin-1 treatment (FIG. 1). Brain edema was alsosignificantly reduced with this combination of rTPA and bryostatin-1(FIG. 2).

The BBB permeability typically increase prior to the occurrence of edemafollowing focal ischemia, such that edema can be used to measure BBBdisruptions at the site of the ischemic lesion. In addition, thehemorrhage process is involved in the BBB disruption and edema. In oneexperiment, uptake of Evans Blue dye was used to measure BBBpermeability, i.e., disruption, and hemorrhaging in ischemic animalmodels of stroke. FIG. 3 shows that combinations of bryostatin andadministered according to the methods of the present disclosuresignificantly reduced uptake of Evans Blue dye in the ipsilateral andcontralateral cortices.

Lastly, increased transport of sodium across the (BBB) contributes tocerebral edema formation in ischemic stroke. FIG. 4 shows that uptake ofNaF in the ipsilateral and contralateral cortices is also reduced withthe disclosed administration regimens of bryostatin-1 and rTPA.

The foregoing results demonstrate that the combination of bryostatin-1with rTPA following ischemic stroke unexpectedly and significantlyreduces mortality and brain injury following ischemic stroke.

Example 4 Synthesis of Fatty Acid Methyl Esters Cyclopropanated FattyAcid Methyl Esters

Synthesis of cyclopropanated fatty acids. Methyl esters of PUFAs werecyclopropanated using the modified Simmons-Smith reaction usingchloroiodomethane and diethylzinc. Tanaka et al., Bioorg. Med. Chem.Lett. (2003), vol. 13, pp. 1037-1040; Furukawa et al., Tetrahedron(1968), vol. 24, pp. 53-58; Denmark et al., J. Org. Chem. (1991), vol.56, pp. 6974-6981. All apparatus were baked at 60° C. for 1 hr and driedusing a flame with dry nitrogen. A 100 ml 3-neck round bottom flask witha stirring bar and a temperature probe was surrounded by an ice-dry icemixture and filled with 1.25 g (4.24 mmol) linoleic acid methyl ester ordocosahexaenoic acid methyl ester in 25 ml dichloromethane and bubbledwith N₂. A 1M solution of diethylzinc (51 ml, 54.94 mmol) hexane wasadded anaerobically using a 24-inch-long 20-gauge needle and thesolution was cooled to −5° C. Diiodomethane (8.2 ml, 101.88 mmol) orchloroiodomethane (ClCH₂I) was added dropwise, one drop per second, withconstant stirring. The rate of addition was decreased if necessary tomaintain the reaction mixture below 2° C. The reaction mixture becamecloudy during the reaction and an insoluble white zinc product wasliberated. The flask was sealed and the mixture was allowed to react for1 hr and then allowed to come to room temperature gradually over 2 hr.

To prevent the formation of an explosive residue in the hood,diethylzinc was not evaporated off. The mixture was slowly poured into100 ml of water under stirring to decompose any excess diethylzinc.Ethane was evolved. The mixture was centrifuged at 5000 rpm in glasscentrifuge tubes and the upper aqueous layer discarded.

The white precipitate was extracted with CH₂Cl₂ and combined with theorganic phase. The organic phase was washed with water and centrifuged.The product was analyzed by silica gel G TLC using hexane plus 1% ethylacetate and purified by chromatography on silica gel using increasingconcentrations of 1-10% ethyl acetate in n-hexane and evaporated undernitrogen, leaving the methyl ester as a colorless oil.

The Simmons-Smith reaction preserves the stereochemistry of the startingmaterials. Furukawa et al., Tetrahedron (1968), vol. 24, pp. 53-58.Docosahexaenoic acid methyl ester was converted into DHA-CP6 in 90-95%yield. The product was a colorless oil with a single absorbance maximumat 202 nm in ethanol and no reaction with I₂. The IR spectrum showedcyclopropane ring absorption at 3070 cm⁻¹ and 1450 cm⁻¹. Under the sameconditions, eicosapentaenoic acid methyl ester was converted to EPA-CP5,and arachidonic acid methyl ester was converted to AA-CP4. Linoleic acidmethyl ester was converted to DCP-LA methyl ester which was identical toa known sample.

Hydrolysis of methyl ester. The methyl ester (0.15 g) was dissolved in 1ml 1N LiOH and 1 ml dioxane. Dioxane and methanol were added until itbecame homogeneous and the solution was stirred at 60° C. overnight. Theproduct was extracted in CH₂Cl₂ and centrifuged. The aqueous layer andwhite interface were re-extracted with water and washed until the whitelayer no longer formed. The product was evaporated under N₂ and purifiedby chromatography on silica gel. The product, a colorless oil, eluted in20% EtOAc in n-hexane. Its purity was checked by TLC in 10% EtOAc/hexaneand by C18 RP-HPLC using UV detection at 205 nm.

The epoxide groups can be introduced by conventional means, e.g., byoxidation of the appropriate alkene with m-chloroperbenzoic acid ort-butylhydroperoxide. Other compounds synthesized include those shown inFIG. 5 (BR-101 through BR-118).

Example 5 Activation of Purified PKCε Using Docosahexanoic Acid

PKC assay. Recombinant PKC (1 ng of PKCα or PKCε isoform) was mixed withBR-101 (DCP-LA) in the presence of 10 micromolar histones, 5 mM CaCl₂,1.2 μg/μl phosphatidyl-L-serine, 0.18 μg/μl 1,2-dioctanoyl-sn-glycerol(DAG), 10 mM MgCl₂, 20 mM HEPES (pH 7.4), 0.8 mM EDTA, 4 mM EGTA, 4%glycerol, 8 μg/ml aprotinin, 8 μg/ml leupeptin, and 2 mM benzamidine.0.5 micro Ci[^(γ32)P]ATP was added. The incubation mixture was incubatedfor 15 min at 37 degrees in a total volume of 10 microliters. Thereaction was stopped by spotting the reaction mixtures on 1×2 cm stripsof cellulose phosphate paper (Whatman P81) and immediately washing twicefor 1 hr in 0.5% H₃PO₄. The cellulose phosphate strips were counted in ascintillation counter. In some experiments, phosphatidylserine,diacylglycerol, and/or calcium were removed.

DHA methyl ester was purchased from Cayman Chemical (Ann Arbor, Me.).PKC isozymes were from Calbiochem (San Diego, Calif.). Purified PKCε waspurchased from Calbiochem.

Results

PKC measurements using purified PKCε showed that, at the lowestconcentration tested (10 nM), compound BR-101 produced a 2.75-foldactivation of PKCε (FIG. 6). PKCα was not affected (data not shown).Compound BR-102 also selectively elicited activation of PKCε to about1.75 fold over unactivated PKCε. The effectiveness of these compounds inactivating PKCε at low concentrations suggests that they will be goodtherapeutic candidates.

Example 6 Activation of Purified or Cellular PKC Epsilon Using Other PKCActivators

Materials. Culture media were obtained from K-D Medical (Columbia, Md.)or Invitrogen (Carlsbad, Calif.). Aβ1-42 was purchased from Anaspec (SanJose, Calif.). Polyunsaturated fatty acid methyl esters were obtainedfrom Cayman Chemicals, Ann Arbor, Mich. Other chemicals were obtainedfrom Sigma-Aldrich Chemical Co. (St. Louis, Mo.). PKC isozymes were fromCalbiochem (San Diego, Calif.). Purified PKCε was purchased fromCalbiochem.

Cell culture. Rat hippocampal H19-7/IGF-IR cells (ATCC, Manassas, Va.)were plated onto poly-L-lysine coated plates and grown at 35° C. inDMEM/10% FCS for several days until about 50% coverage was obtained. Thecells were then induced to differentiate into a neuronal phenotype byreplacing the medium with 5 ml N₂ medium containing 10 ng/ml basicfibroblast growth factor at 39° C. and grown in T-75 flasks at 37° C.Human SH-SY5Y neuroblastoma cells (ATCC) were cultured in 45% F12K/45%MEM/10% FCS. Mouse N2A neuroblastoma cells were cultured in DMEM/10% FCSwithout glutamine. Rat hippocampal neurons from 18-day-old embryonicSprague Dawley rat brains were plated on 12- or 96-well plates coatedwith poly-D-lysine (Sigma-Aldrich, St. Louis, Mo.) in B-27 neurobasalmedium containing 0.5 mM glutamine and 25 μM glutamate (Invitrogen,Carlsbad, Calif.) and cultured for three days in the medium withoutglutamate. The neuronal cells were grown under 5% CO₂ in an incubatormaintained at 37° C. for 14 days.

All experiments on cultured cells were carried out in triplicate unlessotherwise stated. All data points are displayed as mean±SE. BR-101(DCP-LA) was used as its free acid all experiments, while BR-111(DHA-CP6), BR-114 (EPA-CP5), and BR-116 (AA-CP4) were used as theirmethyl esters.

Protein kinase C assay. Rat hippocampal cells were cultured and scrapedin 0.2 ml homogenization buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaF, 1μg/ml leupeptin, and 0.1 mM PMSF) and homogenized by sonication aMarsonix microprobe sonicator (5 sec, 10W). To measure PKC, 10 μl ofcell homogenate or purified PKC isozyme (purchased from Calbiochem) wasincubated for 15 min at 37° C. in the presence of 10 μM histones, 4.89mM CaCl₂, 1.2 μg/μl phosphatidyl-L-serine, 0.18 μg/μl1,2-dioctanoyl-sn-glycerol, 10 mM MgCl₂, 20 mM HEPES (pH 7.4), 0.8 mMEDTA, 4 mM EGTA, 4% glycerol, 8 μg/ml aprotinin, 8 μg/ml leupeptin, and2 mM benzamidine. 0.5 Ci[^(γ32)P]ATP was added and ³²P-phosphoproteinformation was measured by adsorption onto phosphocellulose as describedpreviously. Nelson and Alkon, J. Neurochemistry (1995), vol. 65, pp.2350-2357. For measurements of activation by BR-101 (DCP-LA) and similarcompounds. PKC activity was measured in the absence of diacylglyceroland phosphatidylserine and PKC δ, ε, η, and μ were measured in theabsence of added EGTA and CaCl₂, as described by Kanno et al. (J. LipidRes. 2006, vol. 47, pp. 1146-1150). Low concentrations of Ca²⁺ are usedbecause high Ca²⁺ interacts with the PKC phosphatidylserine binding siteand prevents activation. For measurements of bryostatin activation,1,2-diacylglycerol was omitted unless otherwise stated.

Results and Discussion

To determine their PKC isozyme specificity, fatty acid derivatives werepreincubated with purified PKC for five minutes and the PKC activity wasmeasured radiometrically. As shown for Example 5, above, BR-101 (DCP-LA)was an effective activator of PKCε at 10 μM but had relatively smalleffects on the other PKC isoforms (data not shown). At higherconcentrations BR-101 (DCP-LA) partially inhibited PKCδ (about 1-100 μM)and activated PKCγ (50-100 μM) (data not shown).

BR-111 (DHA-CP6), BR-114 (EPA-CP5), and BR-115 (AA-CP4), thecyclopropanated derivatives of docosahexaenoic acid, eicosapentaenoicacid, and arachidonic acid, respectively, activated purified PKCε to asimilar extent (FIG. 7) The concentration needed to activate PKC wasapproximately 100 times lower than for BR-101 (DCP-LA), suggestinghigher affinity. Cyclopropanated linolenyl and linoleyl alcohols (BR-104and BR-105), epoxystearic acid (BR-116), and vemolic acid methyl ester(BR-117) had little or no effect on PKC (FIG. 8). Cyclopropanatedvemolic acid methyl ester (BR-109) inhibited PKCε at concentrationsabove 1 μM (FIG. 8).

PKC activators that bind to the diacylglycerol binding site, includingbryostatin, gnidimacrin, and phorbol esters, produce a transientactivation of PKC activity, followed by a prolonged downregulation.Nelson et al., Trends in Biochem. Sci. (2009), vol. 34, pp. 136-145.This was confirmed in cultured rat hippocampal cells. Incubation of ratH19-7/IGF-IR cells with (0.04 nM and 0.2 nM) bryostatin produced a2-fold activation that lasted 30 min, followed by a 20% downregulationthat returned to baseline by 24 hours (data not shown). In contrast, PKCexposed to DCP-LA remained elevated for at least four hours (FIG. 9).This sustained activation was only observed in primary neurons.

Even though bryostatin has a higher affinity for PKC than phorbol12-myristate 13-acetate (PMA) (EC50=1.35 nM vs. 10 nM), bryostatin wasmuch less effective than PMA at downregulating PKC. PKC activity isstrongly downregulated by phorbol ester at 8 hours, while PKC inbryostatin-treated cells is at or near the baseline (data not shown).This difference may explain the increases in Aβ produced by PdBureported by da Cruz e Silva et al. J. Neurochem. (2009), vol. 108, pp.319-330. These investigators applied 1 μM PdBu to cultured COS cells for8 hours and observed an increase in Aβ. This increase was attributed todownregulation of PKC by the phorbol ester, which is consistent withthese results. Downregulation could not be measured for DCP-LA andrelated compounds.

Example 7 Effects of PKC Activators on AR Production and Degradation

Cell culture. Cell culture was performed as described in Example 6.

Aβ Measurement and Cell Viability Assay. Aβ was measured using an Aβ1-42 human fluorimetric ELISA kit (Invitrogen) according to themanufacturer's instructions. Results were measured in a Biotek SynergyHT microplate reader. AlamarBlue and CyQuant NF (Invitrogen) accordingto the manufacturer's instructions.

Results and Discussion

To measure the effects of PKCε activation on Aβ production, mouseneuro2a (N2a) neuroblastoma cells transfected with human APPSwe/PSIDwere used, which produce large quantities of Aβ. Petanceska et al., JNeurochem. (1996), vol. 74, pp. 1878-1884. Incubation of these cells for24 hours with various concentrations of PKC activators bryostatin,BR-101 (DCP-LA) and BR-111 (DHA-CP6) markedly reduced the levels of bothintracellular (FIG. 11a ) and secreted (FIG. 11b ) Aβ. With bryostatin,which activates PKC by binding to the diacylglycerol-binding site, theinhibition was biphasic with concentrations of 20 nM or higher producingno net effect. This may be explained by the ability of this class of PKCactivators to downregulate PKC when used at high concentrations. Incontrast, BR-101 (DCP-LA) and BR-111 (DHA-CP6), which bind to PKC'sphosphatidylserine site, showed monotonically increasing inhibition atconcentrations up to 10 μM to 100 μM with no evidence of downregulationat higher concentrations.

To determine whether the reduced levels of Aβ caused by PKC activatorswere due to inhibition of Aβ synthesis or activation of Aβ degradation,BR-111 (DHA-CP6) (0.01 μM to 10 μM) and low concentrations (100 nM) ofexogenous monomeric Aβ-42 were applied to cultured SH-SY5Y cells. Thisconcentration of Aβ is too low to produce measurable toxicity or celldeath. Since SH-SY5Y cells produce only trace amounts of Aβ, thisexperiment was an effective test of the ability of PKC activators toenhance Aβ degradation. By 24 hours, most of the Aβ had been taken up bythe cells and the concentration of Aβ in the culture medium wasundetectable. Addition of 0.01 μM to 10 μM DHA-CP6 to the cells reducedthe cellular levels of Aβ by 45%-63%, indicating that the PKCε activatorincreased the rate of degradation of exogenous Aβ (FIG. 12).

DHA-CP6, bryostatin, and DCP-LA had no effect on cell survival or onproliferation as measured by alamar Blue and CyQuant staining (FIG. 15aand FIG. 15b ), indicating that the reduction in Aβ production did notresult from cell proliferation or a change in cell survival.

Example 8 Effects of PKC Activators on TACE Activity

TACE Assay. TACE was measured by incubating 5 μl cell homogenate, 3 μlbuffer (50 mM Tris-HCl 7.4 plus 25 mM NaCl plus 4% glycerol), and 1 μlof 100 μM TACE substrate (Aβz-LAQAVRSSSR-DPa) (Calbiochem) for 20 min at37° C. 1.5-ml polypropylene centrifuge tubes. Jin et al., Anal. Biochem.(2002), vol. 302, pp. 269-275. The reaction was stopped by cooling to 4°C. The samples were diluted to 1 ml and the fluorescence was rapidlymeasured (ex=320 nm, em=420 nm) in a Spex Fluorolog 2spectrofluorometer.

Results and Discussion

Previous researchers reported that PKC activators such as phorbol12-myristate 13-acetate produce large increases in TACE activity whichcorrelated with increased sAPPα and decreased Aβ, suggesting that TACEand BACE1 compete for availability of APP substrate, and that PKCactivators shift the competition in favor of TACE. Buxbaum et al., JBiol. Chem. (1998), vol. 273, pp. 27765-27767; Etcheberrigaray et al.,Proc. Natl. Acad. Sci. USA (2006), vol. 103, pp. 8215-8220. However,many of these earlier studies were carried out in fibroblasts and othernon-neuronal cell types, which appear to respond differently to PKCactivators than neurons. For example, Etcheberrigaray et al. found thatactivation of PKC in human fibroblasts by 10 pM to 100 pM bryostatinincreased the initial rate of α-secretase activity by 16-fold and132-fold, respectively. However, in human SH-SY5Y neuroblastoma cells,N2a mouse neuroblastoma cells (FIG. 13a ), and primary neurons from rathippocampus (FIG. 13b and FIG. 13c ), PKC activators bryostatin, BR-101(DCP-LA) and/or BR-111 (DHA-CP6) only produced small increases inactivity. This suggests that any reduction of Aβ levels in neurons byPKC activators must be caused by some other mechanism besides activationof TACE.

Example 9 Effects of PKC Activators on Endothelin-Converting Enzyme(ECE) Activity

ECE assay. SH-S757 neuroblastoma cells were incubated with bryostatin(0.27 nM), BR-101 (DCP-LA) (1 μM), and BR-111 (DHA-CP6) (1 μM).Endothelin-converting enzyme (ECE) was measured fluorimetrically usingthe method of Johnson and Ahn (Anal. Biochem. (2000), vol. 286, pp.112-118). A sample of cell homogenate (20 μl) was incubated in 50 mMMES-KOH, pH 6.0, 0.01% C12E10 (polyoxyethylene-10-lauryl ether), and 15μM McaBK2 (7-Methoxycoumarin-4-acetyl[Ala7-(2,4-Dinitrophenyl)Lys9]-bradykinin trifluoroacetate salt)(Sigma-Aldrich). After 60 min at 37° C., the reaction was quenched byadding trifluoroacetic acid to 0.5%. The sample was diluted to 1.4 mlwith water and the fluorescence was measured at ex=334 nm, em=398 nm.

Results and Discussion

Aβ can be degraded in vivo by a number of enzymes, including insulindegrading enzyme (insulysin), neprilysin, and ECE. PKCε overexpressionhas been reported to activate ECE. Choi et al., Proc. Natl. Acad. Sci.USA (2006), vol. 103, pp. 8215-8220. Thus, the effect of fatty acidderivative PKC activators on ECE was examined. Bryostatin, BR-101(DCP-LA), and BR-111 (DHA-CP6) all produced a sustained increase in ECEactivity (FIG. 14). Since ECE does not possess a diacylglycerol-bindingC1 domain, this suggests that the activation by bryostatin was not dueto direct activation of ECE, but must have resulted from phosphorylationof ECE or some ECE-activating intermediate by PKC. This result alsosuggests that indirect activation ECE by PKC activators could be auseful means of reducing the levels of Aβ in patients.

An advantage of compounds that specifically activate PKCε is that theymay produce less downregulation than phorbol esters and similar1,2-diacylglycerol (DAG) analogues. The biphasic response of PKC toDAG-based activators means that a PKC activator may reduce Aβ levels atone time point and increase them at another. Da Cruz e Silva et al., J.Neurochem. (2009), vol. 108, pp. 319-330. Careful dosing and monitoringof patients would be required to avoid effects opposite to thoseintended. The relative inability of compounds to downregulate PKC, suchas the fatty acid derivatives disclosed herein, avoids such unintendedeffects.

Example 10 Global Ischemia Model of Stroke

Rats (male, Wistar, 200-225 g) were randomly divided into 6 groups (8each) and housed for 1 week before experimentation. Transient orpermanent restriction of cerebral blood flow and oxygen supply resultsin ischemic stroke. The global ischemia model used to induce vascularmemory impairment was two-vessel occlusion combined with a short termsystemic hypoxia. Ligation of the bilateral common carotid arteries wasperformed under anesthesia (pentobarbital, 60 mg/kg, i.p.). After aone-week recovery from the surgery, rats were exposed to 14-min hypoxia(5% oxygen in a glass jar). Control rats (sham operated and vehiclecontrols) were subjected to the same incision to isolate both commoncarotid arteries and to 14-min air (in the glass jar). Body temperaturewas kept at 37-37.5° C. using a heating light source during the surgicalprocedure and until the animals were fully recovered.

Example 11 Bryostatin and MCDA Treatment

Bryostatin-1 was administered at 20) μg/m² (tail i.v., 2 doses/week, for10 doses), starting 24 hours after the end of the hypoxic event.4-Methylcatechol-diacetic acid (MCDA, a potential NGF and BDNF booster)was administered at 1.0 mg/kg (i.p., daily for the same 5-week period)in separate groups of rats.

One week after the last bryostatin-1, MCDA, or vehicle administration,rats were. trained in the water maze spatial learning task (2 trainingtrials per day for 4 days), followed by a probe test. A visible platformtest was given after the probe test. The results are shown in FIG. 16.

Overall, there was a significant learning difference between the 6groups (FIG. 16; F_(5,383)=27.480, p<0.001; ANOVA). Detailed analysisrevealed that the ischemic group did not learn the spatial maze tasksince there was no significant difference in escape latency over trials(F_(7,63)=0.102, P>0.05), a significantly impaired learning as comparedwith the control rats (group difference: F_(1,127)=79.751, p<0.001),while the rats in the other 5 groups all learned the task (the ischemicrats with MCDA treatment: p<0.05 and the other 4 groups: p<0.00 lovertrials). Bryostatin-1 therapy greatly improved the performance (Ischemicgroup with bryostatin-1 treatment vs. ischemic rats: F_(1,127)=72.782,p<0.001), to the level of performance that did not differ statisticallyfrom the control rats (Ischemic group with bryostatin-1 treatment vs.control rats: F_(1,127)=0.001, p>0.05). MCDA treatment also improved thelearning of the ischemic rats (ischemia with NCDA treatment vs. ischemicrats: F_(1,127)=15.584, p<0.001) but the difference between the ischemiawith MCDA treatment and control rats remained significant after the 5week treatment (ischemia with NCDA treatment vs. control rats:F_(1,127)=16.618, p<0.001). There were no differences between thecontrol and bryostatin-1-only groups (bryostatin-1 vs. control:F_(1,127)=0.010, p>0.05) and between the control and MCDA-only groups(MCDA vs. control: F_(1,127)=0.272, p>0.05).

The rats in the ischemic group did not show a target preference in theprobe test (F3,31=0.096, p>0.05), while the rats of the other 5 groupsall showed a target quadrant preference in the probe test (all p<0.005).Data were analyzed using target quadrant ratio (dividing the targetquadrant distance by the average of the non-target quadrant valuesduring the probe test; FIG. 17). There was a significant difference inthe target quadrant ratios between the groups (F5,47=5.081, p<0.001).Detailed analysis revealed group differences between the control andischemic rats (F_(1,15)=9.451, p<0.01), between the ischemic andischemic with bryostatin-1 treatment (F_(1,15)=10.328, p<0.01), andbetween the ischemic with MCDA treatment and ischemic rats(F_(1,15)=5.623, p<0.05), but no differences between the control andischemic rats with bryostatin-1 treatment (F_(1,15)=0.013, p>0.05),between the ischemic with MCDA treatment and control groups(F_(1,15)=2.997, p>0.05), between the control and bryostatin-1-only rats(F_(1,15)=0.064, p>0.05), and between the control and the MCDA-only rats(F_(1,15)=0.0392, p>0.05). A visible platform test, determined after theprobe test revealed no significant difference between the groups(F_(5,47)=0.115, p>0.05), indicating that there were no significantgroup differences in sensorimotor ability of the rats.

Example 12 Bryostatin Treatment

Global cerebral ischemialhypoxia was induced in male Wistar rats(225-250 g) by permanently occluding the bilateral common carotidarteries, combined with about 14 minutes of low oxygen (about 5%).Bryostatin-1 was administered at 15 μg/m² (via a tail vein, 2doses/week, for 10 doses), starting about 24 hours after the end of theischemic/hypoxic event. Spatial learning (2 trials/day for 4 days) andmemory (a probe test of 1 minute, 24 hours after the last trial) taskwas performed 9 days after the last dose. Overall, there was asignificant difference between the groups (F3,255=31.856, p<0.001) andgroups x trials (F21,255=1.648, p<0.05). Global cerebral ischemiaimpaired the spatial learning (ischemial vs. sham-operatedF_(1,127)=79.751, p>0.001). The learning impairment was restored byBryostatin-1 treatment (Bryostatin-1+Ischemia vs. Ischemia:F1,127=50.233, p<0.001), while Bryostatin-1 alone did not affect thelearning (Bryostatin-1 vs. sham-operated: F1,127=2.258, p>0.05; 9 daysafter the last dose).

In the memory retention test, sham-operated rats showed a targetquadrant preference. Such good memory retention was not observed in theischemic rats, indicating an impaired spatial memory. Bryostatin-1therapy effectively restored memory retention after ischemia to thelevel of the sham-operated rats. Bryostatin-1 alone had no significanteffects in the target quadrant preference compared with that of thesham-operated control rats. There was a significant difference in thequadrant ratios (calculated by dividing the target quadrant swimdistance by the average swim distance in the non-target quadrants;F3,31=6.181, p<0.005) between the groups. Detailed analysis revealedsignificant differences between the ischemic rats and sham-operatedcontrol rats (F1,15=9.451, p<0.001), between the ischemic rats andischemic rats with Bryostatin-1 treatment (F1,15=10.328, p<0.001), butno significant differences between the ischemic rats with Bryostatin-1treatment and sham-operated control (F1,15=0.0131, p>0.05) and betweenthe sham-operated control rats and Bryostatin-1 alone rats (F1,15=0.161,p>0.05). These results demonstrate that the cerebral ischemic/hypoxiaproduced an impairment of spatial learning and memory, tested about 7weeks after the ischemic event. The impairment was lasting and notrecoverable, during the time frame without appropriate intervention, butrestored by chronic Bryostatin-1 treatment, even when the treatment wasstarted 24 hours after the ischemic event, a wide therapeutic timewindow.

What is claimed:
 1. A method of treating a subject who has suffered anischemic event comprising: (a) administering to the subject ananticoagulant and a protein kinase C (PKC) activator within about 24hours after the ischemic event; and (b) administering at least one PKCactivator after step (a) for a duration of treatment; wherein the PKCactivators of step (a) and step (b) are the same or different.
 2. Themethod of claim 1, wherein the anticoagulant is tissue plasminogenactivator (TPA).
 3. The method of claim 1, wherein the PKC activators ofstep (a) and step (b) each independently bind to at least one of the1,2-diacylglycerol (DAG) and 1,2-diacyl-sn-glycero-3-phospho-L-serine(phosphatidyl-L-serine, PS) sites of PKC, or indirectly activate PKC. 4.The method of claim 1, wherein the PKC activators of step (a) and step(b) are each independently chosen from macrocyclic lactones,diacylglycerol derivatives other than phorbol esters, isoprenoids,daphnane-type diterpenes, bicyclic triterpenoids,naphthalenesulfonamides, diacylglycerol kinase inhibitors, growth factoractivators, and fatty acids and derivatives thereof.
 5. The method ofclaim 4, wherein the macrocyclic lactones are chosen from bryostatin,bryologs, and neristatin.
 6. The method of claim 5, wherein thebryostatin is bryostatin-1.
 7. The method of claim 1, wherein in step(a) the anticoagulant is administered before the PKC activator.
 8. Themethod of claim 7, wherein in step (a) the anticoagulant is administeredwithin 24 hours after the ischemic event.
 9. The method of claim 8,wherein in step (a) the anticoagulant is administered from about 1 hourto about 12 hours after the ischemic event.
 10. The method of claim 9,wherein in step (a) the anticoagulant is administered from about 2 hoursto about 6 hours after the ischemic event.
 11. The method of claim 7,wherein in step (a) the PKC activator is administered within 24 hoursafter administration of the anticoagulant.
 12. The method of claim 11,wherein in step (a) the PKC activator is administered from about 1 hourto about 12 hours after administration of the anticoagulant.
 13. Themethod of claim 12, wherein in step (a) the PKC activator isadministered from about 2 hours to about 6 hours after theanticoagulant.
 14. The method of claim 7, wherein in step (a) theanticoagulant is administered within about 6 hours after the ischemicevent and the PKC activator is administered within about 2 hours afterthe anticoagulant.
 15. The method of claim 14, wherein in step (a) theanticoagulant is administered about 3 hours after the ischemic event andthe PKC activator is administered about 2 hours after the anticoagulant.16. The method of claim 1, wherein in step (a) the PKC activator isadministered before the anticoagulant.
 17. The method of claim 16,wherein in step (a) the PKC activator is administered within 24 hoursafter the ischemic event.
 18. The method of claim 17, wherein in step(a) the PKC activator is administered from about 1 hour to about 12hours after the ischemic event.
 19. The method of claim 18, wherein instep (a) the PKC activator is administered from about 2 hours to about 6hours after the ischemic event.
 20. The method of claim 16, where in instep (a) the anticoagulant is administered within 24 hours afteradministration of the PKC activator.
 21. The method of claim 20, whereinin step (a) the anticoagulant is administered from about 1 hour to about12 hours after administration of the PKC activator.
 22. The method ofclaim 21, wherein in step (a) the anticoagulant is administered fromabout 2 hours to about 6 hours after administration of the PKCactivator.
 23. The method of claim 16, wherein in step (a) the PKCactivator is administered within about 6 hours after the ischemic eventand the anticoagulant is administered within about 2 hours afteradministration of the PKC activator.
 24. The method of claim 23, whereinin step (a) the PKC activator is administered about 3 hours after theischemic event and the anticoagulant is administered about 2 hours afterthe PKC activator.
 25. The method of claim 1, wherein the treatment instep (b) is initiated from about 10 hours to about 32 hours after theischemic event.
 26. The method of claim 25, wherein the treatment ofstep (b) is initiated about 24 hours after the ischemic event.
 27. Themethod of claim 1, wherein in step (b) the PKC activator is administeredfrom 1-3 times per week.
 28. The method of claim 1, wherein the durationof treatment in step (b) ranges from about 1 week to about 10 weeks. 29.The method of claim 1, wherein in step (b) the PKC activator isadministered by intravenous injection.
 30. The method of claim 1,wherein mortality is reduced with respect to administration of theanticoagulant alone.
 31. The method of claim 30, wherein mortality 24hours after the stroke is reduced by at least 40%.
 32. The method ofclaim 1, wherein hemorrhagic transformation is reduced compared toadministration of the anticoagulant alone.
 33. The method of claim 32,wherein the reduction in hemorrhagic transformation is determined bymeasuring the subject's hemoglobin level compared to administration ofthe anticoagulant alone.
 34. The method of claim 33, wherein thehemoglobin level is reduced by about 50%.
 35. The method of claim 1,wherein disruption of the blood-brain barrier is reduced compared toadministration of the anticoagulant alone.
 36. The method of claim 1,wherein the treatment reverses stroke-induced brain injury.
 37. Themethod of claim 1, wherein the treatment reverses stroke-induced memoryimpairment.
 38. A method of treating stroke in a subject in need thereofcomprising: (a) identifying a subject having suffered a stroke; (b)administering to the subject a therapeutically-effective amount of aprotein kinase C (PKC) activator; (c) determining whether the subjectsuffered an ischemic stroke or hemorrhagic stroke; (d) if the subjectsuffered an ischemic stroke, administering a therapeutically-effectiveamount of an anticoagulant; and (e) administering at least one PKCactivator for a duration of treatment; wherein the PKC activators ofstep (b) and step (e) are the same or different.
 39. The method of claim38, wherein step (c) comprises taking a computed tomography (CT) scan.